The long-term objective of this proposal is to understand the molecular mechanisms of vertebrate retinal development and to identify the genes regulating the normal differentiation of retinal neurons. In mice, math5 and brn-3b are expressed early in the developing retinas starting at embryonic day 11 and 11.5 (E11 and E 11.5), respectively. Their temporal and spatial expression pattern in retina correlates with the onset of retinal ganglion cell (RGC) differentiation, suggesting their roles in the differentiation of RGCs. Previous studies have demonstrated that targeted deletion of brn-3b in mice causes the failure of axon formation and the programmed cell death of RGCs in early retinal development. The targeted mutation of math5 results in the absence of a large set of RGCs and abolishes the RGC-specific expression of brn-3b in developing retina. The expression and mutation studies have suggested that math 5 is the proneural gene for the development of RGCs and expression of math5 in the retinal progenitor cells defines the retinal ganglion cell fate. In addition, the absence of brn-3b expression in math5-null retinas suggests that brn-3b is the downstream effector gene of the math5 regulatory pathway in RGC development. Three specific aims are proposed to precisely define the role of math5 in retinal development and to establish relationship between math5 and brn-3b: (1) Mouse lines will be created to activate the stable expression of lacZ reporter gene in math5-expressing cells and the fates of these cells will be analyzed throughout development. Specifically, the effect of math5 mutation on RGC differentiation, migration, programmed cell death or converting to non-RGC fates will be assessed. (2) The ability of math5 to convert the retinal progenitors into RGCs will be tested by expressing math5 in retinal progenitor cells in vivo or in retina explant cultures. (3) The regulatory relationship between brn-3b and math5 will be analyzed using the math5 and brn-3b mutant mice and conventional promoter analysis approaches. Information obtained from these studies will significantly advance our understanding of the roles of transcriptional factors in normal retinal development and in retinal degenerative diseases. Results of these studies could provide the foundation of designing therapeutic approaches to maintain retinal cell viability and to regenerate retinal neurons.